DNA amplification is a process of copying a single or double-stranded target DNA to generate multiple copies of the target DNA. Since DNA strands are antiparallel and complementary, each strand may serve as a template (template strand) for the production of an opposite strand (complementary strand) by a DNA polymerase. The template strand is preserved as a whole or as a truncated portion and the complementary strand is assembled from nucleoside triphosphates. A variety of techniques are currently available for efficient amplification of nucleic acids such as polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), multiple displacement amplification (MDA), or rolling circle amplification (RCA). Many of these techniques generate a large number of amplified products in a short span of time. For example, in a polymerase chain reaction (PCR), a target DNA, a pair of primers and a DNA polymerase are combined and subjected to repeated temperature changes that permit melting, annealing, and elongation steps to result in an exponential amplification of the starting target DNA. However, in PCR, the melting or denaturation step typically occurs at a high temperature limiting the choice of polymerases to thermophilic polymerases.
DNA amplification often suffers from high background signals, which are generated by non-specific amplification reactions yielding undesired/false amplification products. For example, non-specific amplification may result from various primer gymnastics such as nucleic acid template-independent primer-primer interactions. Primers may form primer-dimer structures by intra- or inter-strand primer annealing (intra molecular or inter molecular hybridizations), and may get amplified, and may sometimes predominate, inhibit, or mask the amplification of a target DNA sequence. Such non-specific, background amplification reactions become even more problematic where the target nucleic acid to be amplified is available only in limited quantities (e.g., whole-genome amplification from a single DNA molecule). Efficient DNA amplification techniques are needed, if they are to be used for critical applications such as diagnostic applications, wherein a false-positive amplification may likely result in a wrong diagnosis.
Endonuclease V (also referred as endo V or deoxyinosine 3′ endonuclease) is a DNA repair enzyme that recognizes DNA containing deoxyinosine (a deamination product of deoxyadenosine, also referred as inosine) residues. Endonuclease V primarily cleaves the second or third phosphodiester bond 3′ to an inosine residue in the same strand leaving a nick with a 3′-hydroxyl and a 5′-phosphate. Endonuclease V was first described in Escherichia coli (E. coli). Apart from inosine residues, E. coli endonuclease V also recognizes, to a lesser degree, otherwise modified bases such as abasic sites (AP sites) or urea, base mismatches, insertion/deletion mismatches, hairpin or unpaired loops, flaps and pseudo-Y structures. One or more embodiments of the present invention are directed towards engineered endonuclease V enzymes (mutant endonuclease V) and their use in nucleic acid assays such as strand displacement DNA amplification reactions, wherein the selective nicking capability of mutant endonuclease V is employed to develop an improved method of DNA amplification.